Amalgamating tools of molecular biology, genetics, biochemistry, and immunocytochemistry, this competing continuation application offers an interdisciplinary dissection of key enzymes of the polyamine pathway of Leishmania. The proposal will focus on arginase (ARG), agmatinase (AGM), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), and spermidine synthase (SYN), the entire complement of polyamine and polyamine precursor enzymes in Leishmania. Although our previous investigations were conducted with L. donovani, these studies will be performed primarily with M379 L. mexicana, a strain that can be maintained as promastigotes and both axenic and in vivo amastigotes, thereby enabling our findings to be considered in a more relevant physiological framework. We have isolated L. mexicana DNAs encoding all five of these proteins, sequenced putative full length LmARG and LmAGM PCR products, and cloned and characterized full length LdODC, LdADOMETDC, and LdADOMETDC from L. donovani. In addition, we have generated ldodc, 4ldadometdc, and ldspdsvn knockouts, large and replenishable quantities of LdSPDSYN protein, and mono-specific antisera against LdODC, LdADOMETDC, and LdSPDSYN. These molecular, cellular, biochemical, and immunological reagents are the cornerstone of the three Specific Aims. Multicomponent Specific Aim I focuses on LmARG and LmAGM. LmARG and LmAGM genomic clones will be sequenced and mapped. To assess gene function, we will create lmarg and A/magm null mutants by targeted gene replacement and characterize the resultant phenotypes within both life cycle stages. LmARG and LmAGM will be over-expressed in E. coli, and recombinant protein purified for biochemical studies and raising antibodies. LmARG and LmAGM will then be localized by immunocytochemistry and cell fractionation protocols. The intracellular locations of ODC, ADOMETDC, and SPDSYN will also be assessed. Specific Aim II involves the reconstruction of the odc, adometdc, and spdsyn knockouts in L. mexicana line in order to definitively determine whether polyamine gene function is essential for long term amastigote proliferation, and thus, whether the polyamine pathway offers valid therapeutic targets for treating leishmaniasis Finally, the role of conserved acidic and aromatic amino acids among SPDSYN enzymes in catalytic activity and substrate binding will be defined for the first time by site-directed mutagenesis of LdSPDSYN and biochemical analysis of the mutant ldspdsyn proteins.